Conditions of protection by hypothermia and effects on apoptotic pathways in a rat model of permanent middle cerebral artery occlusion

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Object

Hypothermia is protective in stroke models, but findings from permanent occlusion models are conflicting. In this article the authors induced focal ischemia in rats by permanent distal middle cerebral artery (MCA) occlusion plus transient occlusion of the common carotid arteries (CCAs). This models a scenario in which the MCA remains occluded but partial reperfusion occurs through collateral vessels. The authors also determined whether hypothermia mediates ischemic damage by blocking apoptotic pathways.

Methods

The left MCA was occluded permanently and the CCAs were reopened after 2 hours, leading to partial reperfusion in rats maintained at 37°C, 33°C (mild hypothermia), or 30°C (moderate hypothermia) for 2 hours during and/or after CCA occlusion (that is, for a total of 2 or 4 hours of hypothermia or normothermia). Infarct size was measured 2 days after the stroke. Immunofluorescence staining and Western blot analysis were used to detect cytochrome c and apoptosis inducing factor (AIF) translocation.

Results

Four hours of prolonged mild hypothermia (33°C) reduced the infarct size 22% in the model of permanent MCA occlusion, whereas 2 hours of such mild hypothermia maintained either during CCA occlusion or after CCA release did not attenuate ischemic damage. However, moderate hypothermia (30°C) during CCA occlusion was significantly more protective than 4 hours of 33°C (46% decrease in infarct size). Four hours of mild or moderate hypothermia reduced cytosolic cytochrome c release and both nuclear and cytosolic AIF translocation in the penumbra 2 days after stroke.

Conclusions

These findings suggest that hypothermic neuroprotection might be achieved by blocking AIF and cytochrome c–mediated apoptosis.

Abbreviations used in this paper:AIF = apoptosis inducing factor; CCA = common carotid artery; DAPI = 4′,6-diamidino-2-phenylindole; MCA = middle cerebral artery; PBS = phosphate-buffered saline.

Abstract

Object

Hypothermia is protective in stroke models, but findings from permanent occlusion models are conflicting. In this article the authors induced focal ischemia in rats by permanent distal middle cerebral artery (MCA) occlusion plus transient occlusion of the common carotid arteries (CCAs). This models a scenario in which the MCA remains occluded but partial reperfusion occurs through collateral vessels. The authors also determined whether hypothermia mediates ischemic damage by blocking apoptotic pathways.

Methods

The left MCA was occluded permanently and the CCAs were reopened after 2 hours, leading to partial reperfusion in rats maintained at 37°C, 33°C (mild hypothermia), or 30°C (moderate hypothermia) for 2 hours during and/or after CCA occlusion (that is, for a total of 2 or 4 hours of hypothermia or normothermia). Infarct size was measured 2 days after the stroke. Immunofluorescence staining and Western blot analysis were used to detect cytochrome c and apoptosis inducing factor (AIF) translocation.

Results

Four hours of prolonged mild hypothermia (33°C) reduced the infarct size 22% in the model of permanent MCA occlusion, whereas 2 hours of such mild hypothermia maintained either during CCA occlusion or after CCA release did not attenuate ischemic damage. However, moderate hypothermia (30°C) during CCA occlusion was significantly more protective than 4 hours of 33°C (46% decrease in infarct size). Four hours of mild or moderate hypothermia reduced cytosolic cytochrome c release and both nuclear and cytosolic AIF translocation in the penumbra 2 days after stroke.

Conclusions

These findings suggest that hypothermic neuroprotection might be achieved by blocking AIF and cytochrome c–mediated apoptosis.

Mild hypothermia is a robust neuroprotectant against experimental stroke.13 The protective effects of hypothermia against central nervous system injury and its potential mechanisms have been studied extensively,1,2,6,8 which has led to clinical investigations for patients having suffered a stroke. Despite three recent landmark prospective randomized controlled studies in which mild hypothermia was demonstrated to improve neurological function in patients suffering cardiac arrest from ventricular fibrillation3,11 and to reduce the risk of death or disability in neonates following hypoxic–ischemic encephalopathy,18 additional clinical applications of mild hypothermia for stroke treatment, especially for focal ischemia, should be pursued. To translate hypothermia to the clinical level, it is important to determine whether hypothermia is effective in cases in which recanalization does not totally occur, a phenomenon often observed clinically. However, authors of most prior studies have investigated the protective effect of hypothermia using models with either transient occlusion or complete permanent occlusion, and they have reported that hypothermia is most protective against transient ischemia, although it may have little or no protection at all in models of permanent occlusion. Few studies have addressed the protective effect of hypothermia in an ischemic model with partial reperfusion. Therefore, in this current study we assessed hypothermic neuroprotective effects in a model of focal ischemia produced by permanent distal MCA occlusion plus temporary bilateral CCA occlusion to more closely model this clinical scenario.

Among various protective mechanisms, hypothermia has been shown to reduce ischemic damage by blocking apoptosis. Both caspase-dependent and -independent pathways contribute to apoptotic damage after stroke.9,24,26 In the caspase-dependent pathways, cytochrome c release and caspase-9, -8, and -3 activity have been extensively reported after ischemia.9,16,26 In the caspase-independent pathway, translocation of AIF to the nucleus has been observed after both global and focal ischemia.5,25 We and others have previously shown that hypothermia may block ischemic damage by blocking cytochrome c release or caspase activity after both transient focal and global ischemia;17,22,24 however, whether hypothermia also blocks AIF translocation after stroke is not known. In the current study, we further determined whether hypothermia blocks both cytochrome c and AIF release in a stroke model using permanent MCA occlusion but with partial reperfusion after CCA release.

Materials and Methods

Protocols were approved by the Stanford University Administration on Laboratory Animal Care.

Focal Cerebral Ischemia

Sprague–Dawley rats (290–350 g) were anesthetized with 5% isoflurane and maintained with 2 and 3% isoflurane.25,26 A ventral mid-line incision was made and the two CCAs were isolated. Snares were placed around the CCAs and the animal was placed on its right side. A 2-cm vertical scalp incision was made midway between the left eye and ear. The temporalis muscle was bisected and a 2-mm bur hole was made at the junction of the zygomatic arch and squamous bone. The distal MCA was then exposed and cauterized above the rhinal fissure. The CCA snares were tightened to occlude the CCAs for 2 hours and then were released, while the distal MCA remained occluded.

Animals were divided into six groups (Fig. 1A) and were maintained at 37 °C, 33 °C (mild hypothermia), or 30°C (moderate hypothermia) during and after CCA occlusion. Group 1 was the normothermic control group in which temperature was maintained at 37°C throughout the experiment. In Group 2, mild hypothermia was induced during 2 hours of CCA occlusion but the temperature was increased to 37°C during CCA reperfusion. In Group 3, after 2 hours of normothermia during CCA occlusion, the temperature was decreased to 33°C for 2 hours after CCA release. In Group 4, mild hypothermia (33°C) was maintained for 4 hours during and after CCA release. In Group 5, temperature was further reduced to 30°C during CCA occlusion but increased to 37°C after CCA release. In Group 6, temperature was maintained at 30°C for 4 hours.

Fig. 1.
Fig. 1.

A: Protocols for surgery and temperature management. Six groups of rats were studied. The distal MCA was occluded permanently. The black portion of the bar represents bilateral CCA occlusion (CCAo) for 2 hours and the gray portion indicates 2 hours of temperature management after CCA release (CCAr), including 30°C, 33°C, and 37°C. Rats were allowed to survive for 48 hours after stroke. B: Photographs of representative infarct sections after cerebral ischemia from Groups 1, 4, and 6. Permanent distal MCA occlusion plus 2 hours of bilateral CCA occlusion caused an infarct in the ipsilateral cortex of the occluded MCA (left, Group 1). A coronal section from Level 2 is presented. Four hours of mild hypothermia (center, Group 4) mildly decreased infarct size. When the temperature was reduced to 30°C robust protection was observed (right, Group 6). C: Bar graph showing that hypothermia reduces infarct size after stroke only under certain conditions. A mean infarct size for each group was calculated as the sum of all four levels for each animal divided by the number of animals in each group. The infarct size did not differ among Groups 1 through 3. However, the infarct in Group 4 was reduced about 22% relative to Group 1. When the temperature was decreased to 30°C (Group 5) robust protection was observed; an additional 2 hours of hypothermia in Group 6 did not further reduce infarct size.

Hypothermia was induced by spraying 100% alcohol onto the rat's body. The body temperature was adjusted to 33°C or 30°C 10 minutes before ischemia onset and the temperature was maintained during 2 hours of CCA occlusion. Hypothermia was maintained for another 2 hours after reperfusion, or increased to 37°C for 2 hours (rats remained anesthetized). For postischemic hypothermia, body temperature was decreased to 33°C after CCA reperfusion and maintained for 2 hours (with anesthesia). Temperature was returned to normal with a light and a heating pad after hypothermia. We have previously observed a high correlation between rectal temperature and brain temperature in hypothermic rats;27 therefore, brain temperature was maintained at 33°C, 30°C, or 37°C accordingly. Two days after cerebral ischemia, rats were perfused transcardially with normal saline followed by 4% paraformaldehyde. The brains were postfixed with 4% paraformaldehyde and 20% sucrose for 24 hours, and sectioned into four coronal blocks rostral (Level 1) to caudal (Level 4). Thirty-micrometer sections were cut onto glass slides in the coronal plane using a cryostat. Infarct size was measured at all four levels; a section from each block was selected and stained with cresyl violet. The percentage of infarcted cortex was measured by normalizing to the entire ipsilateral cortex.26

Laser Scanning Confocal Microscopy

Immunofluorescence staining was performed as described.24,26 Tissue prepared as described in the preceding section was also used for this purpose. After washing in PBS, sections were blocked in PBS containing 5% donkey serum (Sigma Chemical Co.), 1% bovine serum albumin, and 0.3% triton X-100 for 1 hour at room temperature and then incubated in the primary antibodies diluted in blocking solution at 4°C overnight. Sections were washed with PBS and incubated for 2 hours at room temperature (light shielded) in the secondary antibodies diluted in blocking solution. Sections were washed with PBS, then incubated in mounting media containing DAPI (Vector Laboratories) for 2 minutes, then washed again with PBS for 10 minutes. Sections were coverslipped and examined under a LSM510 confocal laser scanning microscope (Carl Zeiss). Negative controls, in which the primary antibodies were omitted, were run in parallel.

Primary antibodies of purified goat anti-AIF antibody (dilution 1:200, Santa Cruz) were used. The secondary antibody, horse biotinylated anti–goat 1:200 (Vector Laboratories), was added to tissues and incubated for 2 hours at room temperature followed by washing in PBS 3 × 10 minutes. Streptavidinfluorescein isothiocyanate–conjugated solution (1:200, Molecular Probes) was added and incubated for 30 minutes. After being washed in PBS three times for 10 minutes, the slide was mounted for confocal microscopy examination.

For cytochrome c staining, sections were blocked in PBS containing 5% donkey serum (Sigma), 1% bovine serum albumin, and 0.3% triton X-100 for 1 hour at room temperature. They were then incubated in the primary antibody of purified mouse anti–cytochrome c (1:500, PharMingen) in blocking solution at 4°C overnight. Sections were incubated for 2 hours at room temperature in the secondary antibody of fluorescein isothiocyanate–conjugated donkey anti–mouse immunoglobulin G (1:200, Jackson ImmunoResearch) diluted in blocking solution.

Negative controls, in which the primary antibodies were omitted, were run in parallel.

Western Blot Analysis

Rats that survived 2 days after stroke onset were killed by an overdose of isoflurane and underwent transcardial perfusion with cold PBS. Rat brains subjected to ischemia with normothermia and 2 hours of immediate moderate hypothermia (30°C) after stroke were removed, and tissue corresponding to the ischemic penumbra was dissected for Western blot analysis (Fig. 2A). The ischemic penumbra was defined as the tissue saved by hypothermia 2 days after stroke, and the corresponding region from the normothermic brain or sham animals was dissected for comparison. The fresh brain tissue was used for preparation of subcellular cytosolic and mitochondrial fraction as described.23,25 Western blot analysis was performed as described previously.23,25 Ten micrograms of protein in each lane was loaded and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 4 to 15% Ready Gel (Bio-Rad) for 1.5 hours. Protein bands were transferred from the gel to polyvinylidinene fluoride (Millipore) membranes for 1 hour. Primary antibodies for AIF (1:500, AIF [D-20], Santa Cruz) and cytochrome c (1:1000, Cell Signal) were incubated overnight at 4°C followed by horseradish peroxidase–conjugated secondary anti–goat and anti–mouse antibodies (1:2000), respectively. Protein bands were detected using an enhanced chemiluminescence system (Amersham Biosciences) and exposed to hyper-film. To confirm even loading of protein, membranes were stripped by incubating them in 0.2 M NaOH solution for 5 minutes and probed for β-actin conjugated with horseradish peroxidase (1:40,000, Sigma). To confirm that a pure subcellular cytosolic fraction was obtained from the mitochondria, the mitochondrial marker COX IV antibody was also used to show that the cytosol was not contaminated by the mitochondria.

Fig. 2.
Fig. 2.

A: Schematic indicating regions from where pictures were taken in the second brain section of a rat subjected to nor-mothermic ischemia. Photomicrographs corresponding to the same areas were taken from hypothermic animals. Region I represents an area near the ischemic margin, and Region II is near the ischemic core. Region III from the contralateral nonischemic cortex served as a control. Region IV is the ischemic core, where almost all neurons were killed 2 days after cerebral ischemia. Confocal immunofluoresence microscopy (B–D) showed that hypothermia blocked cytochrome c (Cyto C) release at 2 days after reperfusion. B: Massive cytosolic cytochrome c release was observed in Regions I and II at 2 days in normothermic animals in Group 1. Little positive staining was observed in Region IV (data not shown). No cytochrome c–positive staining was observed in the nonischemic contralateral cortex in Region IV and in negative control in which the primary antibody was omitted (data not shown). C: Prolonged mild hypothermia (33°C for 4 hours, Group 4) attenuated cytochrome c release in Region I but not Region II. D: Two hours of moderate hypothermia (Group 5) not only blocked cytochrome c release in Region I, but attenuated cytochrome c release in Region II as well. Bars = 20 μm.

Statistical Analysis

Two-way analysis of variance was used to compare the protective effect of hypothermia on infarct size followed by the Fisher least significant difference post hoc test. Tests were considered statistically significant at probability values less than 0.05. Data are presented as means ± standard error of the means.

Results

Representative infarcts stained with cresyl violet from Groups 1, 4, and 6 are presented in Fig. 1B. Because the infarcts from Groups 2 and 3 were similar to those in Group 1, and those in Group 5 were similar to those in Group 6, they are not shown. Statistical results for mean infarct size comparison are shown in Fig. 1C. Neither 2 hours of mild hypothermia (33°C) during CCA occlusion (Group 2) nor 2 hours of mild hypothermia (33°C) post-CCA release (Group 3) reduced infarct size compared with normothermic rats (Group 1). Modest protection was observed when 4 hours of prolonged mild hypothermia (Group 4) was used. However, robust protection was achieved when the temperature was dropped to 30°C during 2 hours of CCA occlusion (Group 5), but an additional 2 hours of hypothermia post-CCA release did not provide further protection (Group 6).

A confocal microscopy study was performed to analyze cytochrome c and AIF translocation in the ischemic region topographically 2 days after ischemia onset. A total of four regions were analyzed (Fig. 2). In the normothermic brain, diffusive cytosolic cytochrome c was strongly detected in lesions near the ischemic margin (Regions I and II), but not in the non-ischemic contralateral cortex (Region III). Cytochrome c was not detected in Region IV (data not shown), as most neurons had been lost by 2 days after stroke. Cytochrome c normally expresses in the mitochondria, but it was not detected in the nonischemic cortex (Region III). The lack of staining in Region III may simply reflect that the particular antibody was unable to penetrate into the mitochondria after fixation.26 Thus, the diffusive staining in the ischemic cortex suggests that cytochrome c was released from the mitochondria into the cytosol. Such release was not blocked by 2 hours of mild hypothermia (33°C) maintained either during CCA occlusion or after CCA reperfusion (data not shown), which is consistent with hypothermia not protecting against ischemic damage. However, damage was blocked by prolonged mild hypothermia (4 hours at 33°C) in Region I but not Region II. Moderate hypothermia (30°C for 2 or 4 hours) further attenuated cytochrome c immunostaining in Region II.

Both cytosolic and nuclear AIF translocation were evaluated in normothermic brains (Fig. 3). Like the effect of hypothermia on cytochrome c release, 2 hours of mild hypothermia (33°C) maintained during CCA occlusion or after CCA release did not block AIF translocation (data not shown). However, prolonged mild hypothermia (33°C for 4 hours) blocked AIF release in Region I but not in Region II. Moderate hypothermia (30°C) further attenuated AIF release even in Region II.

Western blot analysis confirmed that moderate hypothermia blocked cytochrome c and AIF release in the penumbra (Fig. 4), where hypothermia spared brain damage.

Fig. 3.
Fig. 3.

Photomicrographs corresponding to regions in the schematic in Fig. 2A. Confocal immunofluoresence microscopy demonstrated that hypothermia blocked AIF translocation at 2 days after reperfusion. A: Nuclear AIF translocation (arrows) after cerebral ischemia was mostly observed in Region I but less in Region II, whereas cytosolic AIF release (arrowheads) was mostly detected in Region II, but less in Region I after ischemia with normothermia (Group 1). No AIF-positive staining was observed in the nonischemic contralateral cortex in Region IV and in negative control in which the primary antibody was omitted (data not shown). B: Prolonged mild hypothermia (33°C) maintained during and post-CCA occlusion (Group 4) blocked AIF nuclear translocation in Region I, but did not effect AIF release in Region II. C: Moderate hypothermia not only blocked AIF translocation in Region I, but also attenuated AIF release in Region II (Group 6). Bars = 20 μm.

Fig. 4.
Fig. 4.

Western blots confirming that hypothermia blocked cytochrome c and AIF release. Both AIF and cytochrome c decreased in the mitochondrial subcellular fraction (left) and concomitantly increased in the cytosolic subcellular fraction (right) after focal ischemia, suggesting their cytosolic translocation from the mitochondria. The COX IV is a mitochondria marker. No COX IV band was detected in the cytosol, indicating pure cytosol was separated from the mitochondria.

Discussion

To facilitate translation of experimental hypothermia to clinical settings, the limitations of its protection must be carefully evaluated so that better strategies can be considered. We found that even intraischemic mild hypothermia (33°C) does not provide protection against permanent MCA occlusion with 2 hours of transient CCA occlusion, suggesting potential limitation of the protective effect of mild hypothermia. Authors of most previous studies agree that hypothermia is neuroprotective against transient ischemia but not permanent ischemia,13 using suture intraluminal MCA occlusion models. Several other investigators used permanent distal MCA occlusion plus ipsilateral CCA occlusion. Two reports among these studies found immediate hypothermia from 30°C to 34.5°C lasting 1 or 24 hours reduced infarct size measured at 1 and 2 days.12,21 Instead, in our current study we used a model in which partial reperfusion was investigated. In one previous study using a similar model of permanent distal MCA occlusion plus 60 minutes of bilateral CCA occlusion, 1 hour of hypothermia (32°C) reduced infarct size measured at 1 day after stroke.15 In the current study, 2 hours of bilateral CCA occlusion was used. To achieve a 22% decrease in infarct size, we had to prolong mild hypothermia (33°C) another 2 hours after CCA release. However, decreasing the temperature from 33°C to 30°C reduced the infarct by 46%. In another study, we found that moderate hypothermia (30°C) reduced the infarct size more than 84% when CCA occlusion was performed for only 1 hour.23 Taken together, in this ischemic model with permanent MCA occlusion plus 2 hours of CCA occlusion, mild hypothermia maintained during ischemia for 2 hours, unless extended to 4 hours, was not neuroprotective. If the temperature was reduced to 30°C or CCA occlusion was reduced to 1 hour, robust neuroprotection was observed. When considering the potential clinical use of hypothermia, a combination of recanalization and hypothermia may be the best therapeutic strategy.

We show for the first time that hypothermia blocks AIF subcellular translocation, in addition to blocking cytochrome c release. Although both caspase/cytochrome c–and AIF-regulated ischemic damage have been studied extensively, authors of only a few reports have addressed the effect of hypothermia on the caspase-dependent apoptotic pathway, and whether hypothermia blocks AIF release is not clear. Mild hypothermia inhibits Fas and caspase-3 expression after 1 hour of focal ischemia.17 Moreover, hypothermia transiently attenuates cytochrome c release but does not alter expression of antiapoptotic protein Bcl-2 or proapoptotic protein Bax in a focal ischemia model.22 However, there was no caspase activity after stroke in that study, suggesting ischemic damage can be caspase-independent, in agreement with findings in other studies.7,10 Most recently, we observed biphasic cytochrome c release after transient global ischemia.24 A small peak of release occurred at 5 hours, and a larger peak occurred at 48 hours after ischemia onset. Caspase-9 and -3 activity significantly increased at 12 and 24 hours following the first phase of cytochrome c release, suggesting that the small amount of cytochrome c might be responsible for the subsequent caspase activity. Although mild hypothermia significantly blocked caspase activity and the second phase of cytochrome c release, it did not block the first phase. This prior experiment suggests that low levels of cytochrome c release might be reversible after global ischemia, which provides a time window for intervention for stroke treatment. Whether early cytochrome c is released in hypothermic brain after focal ischemia needs further investigation. However, we found that moderate hypothermia blocks cytochrome c release in the penumbra 2 days after ischemia onset, suggesting that hypothermia may protect against ischemic damage by blocking the late cytochrome c release.

We have previously shown that AIF translocates into both the cytosol and the nuclei.25 A confocal microscopy study revealed that both cytosolic and nuclear AIF translocation could be blocked by moderate hypothermia. Nuclear AIF binds DNA, causing large DNA fragmentation leading to apoptosis;19 in contrast, the role of cytosolic AIF release is not clear. We have speculated that cytosolic AIF release might be associated more with necrosis occurring after stroke, because cytosolic AIF was mostly detected in the ischemic core where adenosine triphosphate is depleted more severely, making necrosis more likely.25 Nevertheless, both nuclear and cytosolic AIF release were blocked by hypothermia. It is well known that hypothermia has multiple effects against stroke, including delaying anoxic depolarization,20,25 attenuating glutamate release,4 and reducing free radical production.14 Hypothermia may protect against ischemic damage by reducing both cytosolic and nuclear AIF translocation after stroke.

Conclusions

Neuroprotection by hypothermia in a model of permanent MCA occlusion plus transient bilateral CCA occlusion depends on the depth, onset, and duration of hypothermia and the severity of ischemia. Moderate hypothermia protects against ischemia most likely in part by blocking both cytochrome c and AIF subcellular translocation.

Acknowledgment

We thank Beth Hoyte for preparation of the figures and Dr. David Schaal for his support.

References

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    Bernard SAGray TWBuist MDJones BMSilvester WGutteridge G: Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 346:5575632002

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    Corbett DThornhill J: Temperature modulation (hypothermic and hyperthermic conditions) and its influence on histological and behavioral outcomes following cerebral ischemia. Brain Pathol 10:1451522000

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    Gill RSoriano MBlomgren KHagberg HWybrecht RMiss MT: Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain. J Cereb Blood Flow Metab 22:4204302002

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    Mu DChang YSVexler ZSFerriero DM: Hypoxia-inducible factor 1alpha and erythropoietin upregulation with deferoxamine salvage after neonatal stroke. Exp Neurol 195:4074152005

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    Phanithi PBYoshida YSantana ASu MKawamura SYasui N: Mild hypothermia mitigates post-ischemic neuronal death following focal cerebral ischemia in rat brain: immunohistochemical study of Fas, caspase-3 and TUNEL. Neuropathology 20:2732822000

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    Shankaran SLaptook AREhrenkranz RATyson JEMcDonald SADonovan EF: Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J Med 353:157415842005

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    Yanamoto HNagata INiitsu YZhang ZXue JHSakai N: Prolonged mild hypothermia therapy protects the brain against permanent focal ischemia. Stroke 32:2322392001

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This study was supported by National Institute of Neurological Disorders and Stroke Grant Nos. R01 NS27292 (to G.K.S.), R01 NS40516 (to M.A.Y.), and P01 NS37520 (to G.K.S. and R.M.S.).

Article Information

Address reprint requests to: Gary K. Steinberg, M.D., Ph.D., Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive R200, Stanford, California 94305-5327. email: gsteinberg@stanford.edu.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    A: Protocols for surgery and temperature management. Six groups of rats were studied. The distal MCA was occluded permanently. The black portion of the bar represents bilateral CCA occlusion (CCAo) for 2 hours and the gray portion indicates 2 hours of temperature management after CCA release (CCAr), including 30°C, 33°C, and 37°C. Rats were allowed to survive for 48 hours after stroke. B: Photographs of representative infarct sections after cerebral ischemia from Groups 1, 4, and 6. Permanent distal MCA occlusion plus 2 hours of bilateral CCA occlusion caused an infarct in the ipsilateral cortex of the occluded MCA (left, Group 1). A coronal section from Level 2 is presented. Four hours of mild hypothermia (center, Group 4) mildly decreased infarct size. When the temperature was reduced to 30°C robust protection was observed (right, Group 6). C: Bar graph showing that hypothermia reduces infarct size after stroke only under certain conditions. A mean infarct size for each group was calculated as the sum of all four levels for each animal divided by the number of animals in each group. The infarct size did not differ among Groups 1 through 3. However, the infarct in Group 4 was reduced about 22% relative to Group 1. When the temperature was decreased to 30°C (Group 5) robust protection was observed; an additional 2 hours of hypothermia in Group 6 did not further reduce infarct size.

  • View in gallery

    A: Schematic indicating regions from where pictures were taken in the second brain section of a rat subjected to nor-mothermic ischemia. Photomicrographs corresponding to the same areas were taken from hypothermic animals. Region I represents an area near the ischemic margin, and Region II is near the ischemic core. Region III from the contralateral nonischemic cortex served as a control. Region IV is the ischemic core, where almost all neurons were killed 2 days after cerebral ischemia. Confocal immunofluoresence microscopy (B–D) showed that hypothermia blocked cytochrome c (Cyto C) release at 2 days after reperfusion. B: Massive cytosolic cytochrome c release was observed in Regions I and II at 2 days in normothermic animals in Group 1. Little positive staining was observed in Region IV (data not shown). No cytochrome c–positive staining was observed in the nonischemic contralateral cortex in Region IV and in negative control in which the primary antibody was omitted (data not shown). C: Prolonged mild hypothermia (33°C for 4 hours, Group 4) attenuated cytochrome c release in Region I but not Region II. D: Two hours of moderate hypothermia (Group 5) not only blocked cytochrome c release in Region I, but attenuated cytochrome c release in Region II as well. Bars = 20 μm.

  • View in gallery

    Photomicrographs corresponding to regions in the schematic in Fig. 2A. Confocal immunofluoresence microscopy demonstrated that hypothermia blocked AIF translocation at 2 days after reperfusion. A: Nuclear AIF translocation (arrows) after cerebral ischemia was mostly observed in Region I but less in Region II, whereas cytosolic AIF release (arrowheads) was mostly detected in Region II, but less in Region I after ischemia with normothermia (Group 1). No AIF-positive staining was observed in the nonischemic contralateral cortex in Region IV and in negative control in which the primary antibody was omitted (data not shown). B: Prolonged mild hypothermia (33°C) maintained during and post-CCA occlusion (Group 4) blocked AIF nuclear translocation in Region I, but did not effect AIF release in Region II. C: Moderate hypothermia not only blocked AIF translocation in Region I, but also attenuated AIF release in Region II (Group 6). Bars = 20 μm.

  • View in gallery

    Western blots confirming that hypothermia blocked cytochrome c and AIF release. Both AIF and cytochrome c decreased in the mitochondrial subcellular fraction (left) and concomitantly increased in the cytosolic subcellular fraction (right) after focal ischemia, suggesting their cytosolic translocation from the mitochondria. The COX IV is a mitochondria marker. No COX IV band was detected in the cytosol, indicating pure cytosol was separated from the mitochondria.

References

1

Auer RN: Non-pharmacologic (physiologic) neuroprotection in the treatment of brain ischemia. Ann N Y Acad Sci 939:2712822001

2

Bell TEKongable GLSteinberg GK: Mild hypothermia: an alternative to deep hypothermia for achieving neuroprotection. J Cardiovasc Nurs 13:34441998

3

Bernard SAGray TWBuist MDJones BMSilvester WGutteridge G: Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 346:5575632002

4

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